A number of publications exist for various forms of GHRH-coding DNA plasmid constructs including patents for a product currently marketed for veterinary use in Australia under the name Lifetide® by VGX PHARMACEUTICALS, LLC out of Blue Bell, Pa., a company acquired by Inovio Pharmaceuticals of Blue Bell, Pa.
There are two major conventional methods of gene therapy, the first, generally better known method, is to use viral capsids to encapsulate a DNA sequence of interest for introduction into animal tissue. Such viral capsids are generally termed viral vectors, and a variety of vectors have been used including HIV and adenoviruses. Such viral vectors are generally constructed so that they should not reproduce in-vivo, and usually contain a reverse transcriptase that results in splicing of the viral vector borne DNA into the host animal's genome.
The viral vector method has multiple drawbacks, not least of which is that it has caused patient death and serious illness, both short term (e.g. idiopathic apparent immune system storm) and long term (e.g. cancer), despite the relative rarity of this type of gene therapy's use. One patient has also died due to the gene of interest resulting in sufficient immune system suppression that a probably quiescent infection to bloom. Thus, while gene therapy had been perceived as having much promise, there are issues, most of which have little to do with the genes of interest that are introduced.
By contrast, the second major conventional method of gene therapy, DNA gene therapy, uses the much simpler method of injection of DNA into the patient. This introduces considerably less packaging material into the host, and DNA constructs are generally smaller. DNA is not incorporated into the host genome, but is instead maintained separately in circlets either inside the nucleus or at the nuclear wall inside the cell. These circlets may become associated with histones within the nucleus. However, DNA gene therapy has few inherent problems. DNA gene therapy does suffer from difficulty producing enough of the gene product. It also may have a small percentage of subjects who are stimulated to produce an immune response to the protein product of the gene of interest. This effect is due to the self-adjuvant effect of plasmids, which contain gene sequence motifs which are recognized by animal immune systems as having a non-self origin. These motifs contain cytosine followed by guanine in the 5′ to 3′ direction, also called CpG sequences (Brazalot-Millan et al, 1998).
Because GHRH has a half-life after injection of roughly 8 minutes, it has not been practical to supplement using conventional methods. However, GHRH is a peptide hormone, which makes DNA gene therapy a viable alternative for supplementation. In addition, GHRH is a hormone that is required in very small amounts. These three factors together make it an appropriate choice to consider for DNA gene therapy.
Thus, there is a need to improve GHRH gene constructs. The present invention minimizes immune system activation by anti-adjuvant codon optimization to eliminate CpG sequences to prevent binding to bacterial DNA intracellular receptors.
In addition, methods and compositions are provided that further minimize the activation of the immune system by any DNA construct delivered in-vivo using co-delivered compounds (Yu et al, 2008).
The Applicant further provides compositions for improved regulation and expression of the plasmids by means of AT rich regions that are in the range of 20 base pairs to 1000 base pairs long placed 5′ and 3′ to the gene expression cassette.
There are further compositions to improve secretion of the produced protein by providing a leader sequence on the gene which codes for a peptide that signals the cell to transport the gene product to the cell membrane for export. (Zou, 2006)
And finally, there is postproduction modification of plasmids to excise the bacterial components so as to yield a smaller circlet of DNA bearing the expression cassette only. This allows use of higher yield bacterial origins of replication containing large CpG sequences so as to get higher concentration of plasmid in production, while preventing the shutdown of plasmid gene expression that is associated with DNA bearing bacterial sequences.
It is therefore a primary object of the invention to improve suitability of GHRH constructs for human therapeutic use.
It is a further object of the invention to provide methods for minimizing immunogenicity of DNA constructs delivered into cells.
It is a further object of the invention to provide methods for maximizing ongoing expression of DNA constructs delivered into cells.
Other objects and advantages of the present invention will become apparent to the reader and it is intended that these objects and advantages are within the scope of the present invention.
As a practical matter, when expanding cultures of bacteria to reproduce usable quantities of plasmids for gene therapy, minor mutations occur, most of which are of no significance. In this invention, such mutations are only significant when they result in changes that create antigenic responses not created by the designed plasmid or else functional modification of the peptide sequence for the GHRH gene.